Regeneration in sponge Sycon ciliatum mimics postlarval … · 2020. 5. 4. · Sycon cil iatum, a calcareous sponge , is widely explored in embryonic and regeneration studies [8,

Regeneration in sponge Sycon ciliatum mimics postlarval development

Anael Soubigou1,3, Ethan G Ross2,3, Yousef Touhami3, Nathan Chrismas3, Vengamanaidu Modepalli3*

1 Sorbonne University, Faculté de Sciences et Ingénierie, Campus Pierre et Marie Curie Jussieu, Paris 75252, France 2 University of Southampton, School of Ocean and Earth Science, Southampton, SO17 1BJ, United Kingdom 3 Marine Biological Association of the UK, The Laboratory, Citadel Hill, Plymouth PL1 2PB, United Kingdom

*Corresponding author: [email protected]

Abstract

Somatic cells dissociated from an adult sponge can re-organize and develop into a functional

juvenile. However, the extent to which regeneration recapitulates embryonic developmental

signaling pathways has remained enigmatic for more than a century. To this end, we have

standardized and established a sponge Sycon ciliatum regeneration protocol to achieve

consistent regeneration in cell culture. From the morphological analysis, we demonstrated that

dissociated sponge cells follow a series of morphological events resembling embryonic and

postlarval development. Hence, we propose that sponge regeneration represents somatic

development. To support our hypothesis, we performed high-throughput sequencing on

regenerating samples and compared the data with regular embryonic and postlarval

development of Sycon ciliatum. Our comparative transcriptomic analysis illuminates that

sponge regeneration is equally as dynamic as embryogenesis. We find that sponge regeneration

is orchestrated by complex regulatory mechanisms by recruiting signaling pathways like those

utilized in embryonic development to organize into a functional juvenile. In the current study,

we lay down the basic framework to study Sycon ciliatum regeneration. Since sponges are

likely to be the first branch of extant multicellular animal and the sister lineage to nearly all

animals, we suggest that this system can be explored to study the genetic features underlying

the evolution of multicellularity and regeneration.

Introduction

The gradient of regeneration widely differs in the animal kingdom and among them, the

sponge is an exceptional organism with an extraordinary regenerative ability. Anatomical

plasticity and cell differentiation is a characteristic feature of sponges, and many sponge cells

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are capable of transdifferentiation [1, 2]. Dissociated cells from an adult sponge have the

unique ability to reaggregate and fully reconstitute into a functional sponge [3-5]. Despite its

significance, we still lack an in-depth understanding of the molecular mechanisms and cellular

basis of sponge regeneration. Sponges are the ancient extant branch of metazoans (branching

order is still debated) [6] and understanding their cellular behavior or morphogenesis during

regeneration can provide insights into the evolution of multicellularity and regeneration.

Calcareous sponges have been extensively studied in the past centuries and analysis of their

development has significantly influenced evolutionary theory. Terms such as gastrulation and

metamorphosis were first coined in syconoid species from the classic Calcaronea studies [7].

Sycon ciliatum, a calcareous sponge, is widely explored in embryonic and regeneration studies

[8, 9].

In S. ciliatum, embryogenesis is viviparous. In brief, after fertilization the embryos

through early cleavages develop into a rhomboid shape. Subsequent cell divisions result in the

formation of a cup-shaped embryo (stomoblastula). The embryo is composed of granular

macromeres adjacent to the choanocytes and ciliated micromeres pointing into the embryonic

cavity. After inversion, the cilia are positioned on the outer surface of the larva and eventually

the ciliated amphiblastula larva migrates into the radial chamber and swims out of its parent

(Fig.1) [8-10]. The amphiblastula larva is composed of a single layer of embryonic cells:

ciliated micromeres on the anterior pole, and non-ciliated granular macromeres on the posterior

pole. During metamorphosis, the ciliated cells of the anterior half undergo an epithelial-to-

mesenchymal transition and form the inner cell mass; the choanocytes will differentiate from

these cells. The macromeres envelop the inner cell mass and gradually transform into

pinacocytes (pinacoderm). After 24 hours following settlement, the spicules (monaxons) are

produced by sclerocytes which have differentiated from the inner cell mass. A choanocyte

chamber is formed in the inner cell mass and develops into the choanoderm. An osculum is

formed at the apical end and the juvenile is elongated along the apical-basal axis rises into a

syconoid body plan [8, 10] (Fig.1).

Many studies have been dedicated to S. ciliatum regeneration to understand their

morphological signatures, starting from Wilson 1907 [3], the pioneer of this topic. Despite

their strong morphological resemblance to embryonic and postlarval development, to date, no

study has systematically compared them. Several transcriptome studies of regeneration in

cnidarians and bilaterians have demonstrated that many developmental pathways are recruited

in regeneration [11-14]. Hence we aim to study the regeneration of S. ciliatum and compare it

to embryonic and postlarval development at both morphological and transcriptional levels. We



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first carried out S. ciliatum regeneration to acquire morphological data for comparison to

regular embryonic and postlarval development and classified the regeneration into seven key

stages. Then, we collected the regenerating aggregates at defined time points and performed

high-throughput sequencing to compare the transcriptional profile with those of normal

embryonic and postlarval development.

Results and Discussion

Sycon ciliatum regeneration morphologically resembles embryonic and postlarval development.

First, we standardized and established a S. ciliatum regeneration protocol to achieve

consistent sponge regeneration in cell culture. Dissociated cells sink to the bottom of the dishes

within 1 hr of dissociation. Among these cells, we detected choanocytes, amoeboid cells, pieces

of spicules and round cells. The majority of the cells lose their cell morphology. In 4-6 hours

post dissociation (hpd), loose multicellular aggregates of diverse shapes (~10 µm) are formed;

they are known as primmorphs. The contacts between the cells were not strong, and upon

shaking the aggregates fell apart into separated cells. Within 24 hpd, these primmorphs

significantly increased in size (~100 µm) predominantly due to the incorporation of cells from

suspension or by fusing with other aggregates (Fig.2 A-C). The primmorphs have no defined

surface epithelium; on the surface pinacocytes and amoebocytes, as well as flagella and collars

of choanocytes were noticeable and similar types of cells can be found inside the primmorphs

(Supplementary file 1-3). During the early stages the cells are actively reorganized and at 48

hpd most of these cells have lost their typical features, primarily due to transdifferentiation

(Fig.2 J-K). The primmorphs become denser and spherical and two layers of cell types can be

identified: a uniform single-cell layer of pinacocytes on the surface and loosely packed granular

cells inside the primmorphs (Fig.2 D-F) (Supplementary file 3). Earlier studies coined this

phenomenon of forming bilayer cells as redevelopment, or somatic development [5, 15].

Choanocytes on the surface were oriented with their flagella outwards and retained their

characteristic features up to 2-3 days post dissociation (dpd) and after that, they started

gradually losing their typical features (flagellum and microvilli) and becoming

indistinguishable from other cells (Fig.2 I, L). Largely, two types of primmorphs were

observed in the culture: the first one is granulated cell packed spheroid with a small bubble and

another one is with a large bubble and a small inner cell mass (termed as blow-outs by Julian

S. Huxley. [4]), an interesting morphology that resembles a blastula with blastocoel.



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Eventually, a single continuous cavity is formed between the external single cell layer of

pinacocytes and internal cell mass (Fig.2 H). At 3 dpd, the primmorphs were entirely covered

with pinacocytes, which may have possibly formed through transdifferentiation from

choanocytes (Fig.2 J-L).

Around 5 dpd spicules start appearing from the primmorphs and simultaneously multiple

ciliated chambers start appearing inside the cell mass. Similarly to regular postlarval

development [8], the monaxons were formed first (Fig.3 A, D). Over the next 6-10 days, the

ciliated chambers expanded and became lined with choanocyte epithelium (Fig.3 G-I)

(Supplementary file 4 & 5). Gradually, these choanocyte chambers fuse into a single cavity

lined with choanocytes (also known as choanoderm). Around 16 dpd a single extended cavity

(spongocoel) with osculum forms at the apical end, and simultaneously multiple porocytes with

ostia are formed (Fig.4 A, C) (Supplementary file 6). These juveniles continue elongating

along the apical-basal axis, and long straight spicules form a crown around the osculum.

Further development can be described as an increase in the complexity of spongocoel and the

development of intermediate porocytes with ostia (Fig.4 E-J) (Supplementary file 7).

Regeneration in S. ciliatum displayed a multitude of complex morphogenesis steps,

progressing through a series of aggregation, organization, and somatic-development events.

Based on our current observations and multiple pieces of evidence from previous studies [3-

5], we classified sponge regeneration into seven stages based on the morphological and cellular

events: I) Aggregation, II) Primmorphs, III) Primmorphs with monaxons, IV) Primmorphs with

ciliated chambers, V) Choanoderm, VI) Pre-Juvenile (Ascon stage), VII) Juvenile; the details

are presented in the following table (Table 1, 3rd column). Remarkably these morphological

signatures are vastly comparable to embryonic and postlarval development of S. ciliatum (Fig.1

and Table 1, 4th column) [1, 8, 9]. To gain further insights into their morphological overlap,

we compared regeneration with both embryonic and postlarval development. The

embryogenesis of calcaronean sponges was previously well described on light and electron

microscopy levels [1, 8, 9]. The morphological comparison was performed using the data from

the published S. ciliatum embryonic development studies (Leininger, S., et al. 2014 and Eerkes-

Medrano, D. I. and S. P. Leys 2006). As demonstrated in Table 1, apart from initial stages of

primmorphs at day 1, 2 and 3, the majority of S. ciliatum regeneration steps are overlapping

with a normal S. ciliatum development directly after metamorphosis including S2, S3, S4, S5

and YS [8].



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The gene expression of regenerating Sycon ciliatum is as dynamic as postlarval development

A long-standing question in the field of sponge regeneration is whether and to what

degree regeneration recapitulate embryonic developmental pathways. Extensive morphological

similarities between regeneration and postlarval development (PLD) have driven us to explore

the gene expression pattern of both processes. We sequenced regenerating samples at 7-time

points spanning from 1 to 24 days including dpd 1, 2, 3-4, 6-8, 10-14, 16-18 (PJUV) and 21-

24 (JUV). For postlarval development, the RNAseq datasets spanning 7-time points were

collected from previously published data (PRJEB7138 & PRJEB5970) including

amphiblastula (swimming larva), flat post larva (S1), solid post larva (S2), hollow post larvae

(S3), choanocyte chamber (S4 & S5) and asconoid juvenile (PY) stages [8]. For comparative

analysis, we processed both data sets through similar pipelines as described in the material and

methods section. To assess the global transcriptomic profile underlying postlarval development

and regeneration, we performed principal component analysis (PCA) on individual data sets.

We found that both regeneration and PLD follow similar PCA distribution and surprisingly the

transcriptional changes during regeneration are as variant as PLD (Fig.5 A&B). We also

performed hierarchical clustering (HC) on both datasets to define the major clusters and we

identified two major clusters that we classified as early and late stages (Fig.5 C&D). From

PCA and HC analysis we observed that clusters in regeneration are organized in a hierarchy

consistent with what we observed in the PLD.

We then directly compared the transcriptomic variation among regeneration and PLD

using PCA. Despite their transcriptomic dynamics, regeneration and PLD fall apart from each

other mainly at PC1 proportion. However, the profiles of regeneration and PLD began to

converge as they progress through development (Fig.6 A). This indicates that the

transcriptomic dynamics of regeneration and PLD are probably similar at advanced stages.

Similar conclusions were also drawn in HC analysis: the late stages (D10-14, PJUV & JUV)

from regeneration have clustered with S5 and PY from PLD (Fig.6 B&C), further suggesting

that the later stages of both regeneration and PLD may express genes with maximum

correlation. High variance among early time points of regenerating and developmental samples

indicates an alternative path was taken during the early stages of regeneration. As noted from

the morphology, early stages of regeneration are not strictly a morphological recapitulation of

development with potential for extensive temporal plasticity during early regeneration.

A further comparison was carried out on the significantly differentially expressed genes

(padj (FDR) < 0.01) at any time point compared to day1 for regeneration and S1 for PLD.



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Regeneration exhibited 8937 differentially expressed genes (DEG), which is comparable to

9847 DEG in PLD, suggesting that regeneration exhibits dynamic gene expression equivalent

to PLD. Of these dynamically expressed genes, approximately 50% of genes (4463) are

commonly expressed in both data sets (Fig.6 D), suggesting that half of the PLD genes were

deployed during regeneration. As regeneration employs 50% of PLD genes, we intended to

compare the expression profiles of these commonly differentially expressed genes. We

subjected the candidates to Fuzzy c-means clustering [16] to group the genes based on the

expression profile (Fig.7 A&B). The clustered genes were compared between regeneration and

postlarval development. We found that the majority of the regeneration gene clusters exhibited

significant overlap with specific PLD clusters (Fig.7 C&D). We conclude that regeneration

partly resembles PLD by displaying similar gene expression profiles.

Overall, we find that late stages from both regeneration and PLD have a high correlation,

thus the major variation in these data is among the early stages. Since the regeneration is not

starting from a fertilized single-cell embryo or amphiblastula larva, early regeneration stages

do not necessarily need to follow a similar pattern of gene expression profile to normal

embryonic and PLD development. Despite its rudimentary embryonic and swimming larval

morphology, the early primmorphs were not close to normal swimming larva in gene

expression profile, however the subsequent stages do resemble postlarval development (Fig.6).

The early primmorphs are unique, as observed from morphological analysis, and multiple

cellular events at this stage are expected to have unique gene expression.

Analyzing S. ciliatum regeneration gene expression

To the best of our knowledge, sponge regeneration from dissociated somatic cells has

not been examined at the transcriptome level. We analyzed the regeneration gene expression

data to identify developmental and multicellular regulatory genes. We performed a GO-term

enrichment analysis to reveal gene network acting during regeneration. The analysis revealed

that a set of transcription factors tightly controls the regeneration process. A broad range of

eumetazoan developmental regulatory genes were expressed during regeneration, gene families

such as Wnt, Tgfb, Fzd, and Smad were differentially expressed (Fig.8 A). Surprisingly, the

majority of listed genes were previously identified in S. ciliatum during embryonic and

postlarval development [8, 17-19]. From the pathway enrichment analysis we identified Wnt,

TGF-beta, Notch, Hedgehog, FGF and EGF signaling pathways, and the majority of core

pathway components were detected (Fig.8 A, Supplementary file 8). Each of these signaling



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pathways triggers several downstream signaling cascades regulating cell behavior such as cell-

cell and cell-ECM interactions [20]. Along with other biological processes, apoptosis plays an

important role during regeneration [21, 22]. Apoptosis mediated regeneration permits cell

elimination and reorganizes cell composition. Major cell death or apoptotic associated

pathways including Wnt signaling, oxidative stress response and p38 MAPK pathway were

expressed during S. ciliatum regeneration (Supplementary file 8), revealing the importance of

apoptosis in remodeling the primmorphs to initiate re-development.

After tissue dissociation, the initial cellular source constitutes a heterogeneous pool of

adult somatic cells. During the initial regeneration phase (day 1 to 3) the dissociated cells re-

aggregate and organize into a morphologically distinguishable structure (Fig.2). During the

development of multicellular organisms, cells change their position extensively and this

process is governed by cell-adhesion and motility molecules. Similarly, the sponge

regeneration is guided through a series of cellular events including initial cell aggregation and

followed by cell organization. Hence, we analyzed the genes associated with cell adhesion and

cell mobility molecules. Using GO term analyses of DEG, we identified members associated

with cell-adhesion gene families, including those that encode cell-surface receptors,

cytoplasmic linkers, and extracellular-matrix proteins (Fig.8 B). Expression of these cell-cell

and cell-ECM adhesions signify highly integrated networks crucial for the individual cells to

adhere and organize into a multicellular structure [23]. Cadherins mediate cell-cell adhesion

broadly regulating morphological aspects during development [24, 25]. Several cadherin

pathway associated genes were significantly differentially expressed (Fig.8 B). Along with

cadherins we also identified integrin pathway associated genes. Cell migration is a process that

is highly dependent on adhesion and junction molecules such as integrins [26, 27]. The co-

expression of cadherin, integrins and other down-stream scaffolding and adaptor proteins such

as RhoGTPases, tyrosine kinases, and phosphatases, suggests that together these molecules

contribute to initiating the early events of regeneration. Integrins mediate contact between cells

and ECM in many organisms by binding to ECM proteins [28-32]. Three major extracellular

matrix structural proteins fibronectin, collagen and laminin displayed differential expression

(Fig.8 B), demonstrating the importance of ECM molecules during regeneration. In summary,

our analysis indicates that initial phases of regeneration, including cell aggregation and

organization, are guided by a set of cell-cell and cell-ECM adhesion associated molecules.



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Lessons to learn on the origin of multicellularity over the sponge regeneration model.

The factors that have driven the emergence of multicellular animals from their unicellular

ancestors have yet to be addressed. Several studies on unicellular relatives of animals including

choanoflagellates, filastereans, and ichthyosporeans have significantly contributed to

understanding how unicellular organisms transform into multicellular structures such as

colonial choanoflagellate [33-35]. This suggests similar transitions may have taken place in the

animal ancestors, which eventually evolved into a stable and functional multicellular animal

[36, 37]. The current hypothesis on the origin of multicellular animals generally agrees that

choanoflagellates share a common ancestor with animals (Fig.9). This idea is based on the

molecular phylogeny and morphological similarity between choanoflagellates and sponge

choanocytes (Fig.9 A-C) [38]. Further, the choanoflagellate colonies partially resemble the

choanocyte chambers of the sponge [37, 39, 40]. In addition to their unicellular sister lineages,

we also need to investigate other close relatives of the first multicellular animals.

Studying the early regeneration events in sponge, including the signalling molecules

triggering the aggregation of cells, can provide a window into understanding the transition from

unicellular to multicellular life forms. Recent studies on multiple unicellular holozoans

revealed several genes related to multicellularity including cell differentiation, cell-cell, and

cell-ECM adhesion (Fig.9 D) [41-44], suggesting these factors have evolved before the origin

of multicellular animals. Indeed, in our current analysis on S. ciliatum regeneration gene

expression, we found that several cell signalling and adhesion protein families including

cadherin, integrins, and ECM molecules were highly expressed within 24 hrs from dissociation

(Fig.8 B). The main focus of the current study is to test the homology between regeneration

and embryonic development, hence sampling was prioritized based on morphology with the

first sample collected 24 hr following dissociation. To gain an in-depth understanding of the

initial signalling cascades triggering the aggregation a tight sampling during the initial

aggregation stages would be beneficial. Future studies in this direction will provide further

insights into the evolutionary mechanisms underlying the origin of multicellular animals.

Conclusions

Sponge regeneration is a powerful system to understand the cellular and molecular mechanisms

governing animal regeneration in general. This is crucial for understanding the evolution of

multicellularity. We compared regeneration and embryonic development in S. ciliatum by

applying both morphological and RNA-seq analyses to reveal the correlation among these



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phenomenons. Here we demonstrated that S. ciliatum regeneration is governed by core

components of the Wnt, Tgfβ, Notch and Hedgehog signaling pathways, which are known to

guide various aspects of embryonic development and morphogenesis in extant eumetazoans.

Furthermore, we have identified major eumetazoan cell-adhesion gene families, including

those that encode cell-surface receptors, cytoplasmic linkers, and extracellular-matrix proteins.

We found that approximately 50% of genes differentially expressed during postlarval

development are also expressed in regeneration. The S. ciliatum dissociated cells thus deploy

a similar set of genes as their embryonic and larval development to assist regeneration in cell

culture. This has important implications for using the S. ciliatum regeneration system to model

the evolution of multicellularity. In the current study our sampling was aimed to dissect the

early cellular events in regeneration, an additional high throughput studies over multiple time

points can address deeper questions such as somatic cell trajectory during regeneration.

Methods

Animal collection and cell culture

Adult S. ciliatum specimens were collected in the vicinity of Plymouth, Devon, UK. All

specimens were at the stage of the post reproduction growth and did not contain any

reproductive elements. Prior to experimentation, animals were maintained in the aquarium with

filtered seawater (FSW) no longer than 2-4 hr. Each S. ciliatum was cut into small pieces by

using a scalpel and micro scissors, then mechanically passed through 40 µm nylon mesh

(Fisherbrand™) into a petri dish (35 mm). The dissociated cells were suspended in FSW and

pipetted multiple times to allow maximum dissociation. The dissociated cells from each

individual were distributed into six-well plates. The number of cells in the plates ranged from

1.2×106 -1.5×107. Cell cultures were maintained at 15°C in dark, every 24 hr the culture

medium was exchanged with FSW. The cell culture was observed daily, and bright-field

images were collected. Initially, when attempting to follow published protocols [3-5], we were

unsuccessful with the S. ciliatum regeneration, the majority of the cell aggregates were unable

to progress to functional juveniles. Hence, we modified the protocol by fractionating the

dissociated cells through centrifugation. We followed similar instructions until cell

dissociation, and then we centrifuged the dissociated cell at 1.2 rpm for 2 min. The supernatant

was collected into a new 1.5 ml tube and re-centrifuged at 2.5 rpm for 2 min. The cell pellets

from both steps were suspended in FSW and distributed individually in 6 well plates. The cells

pelleted at 2.5 rpm have a high regeneration ability in comparison to the cells pelleted at 1.2



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rpm. In the current study, we used the aggregates collected from 2.5 rpm wells. Apart from

standardizing the protocol, the regeneration experiment was performed 7 times independently

and 8 to 12 specimens were used in each experiment. For morphological analysis, the samples

were collected on days post dissociation 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 18, 21 and 24. 10-20

regenerative structures were collected per replicate.

Whole-mount immunofluorescence

The specimens were fixed for 1 hr at 4°C on a roller with 0.05% glutaraldehyde and 4%

paraformaldehyde (PFA) in PBS buffer. After fixation, the samples were washed 5 times with

PBST (1× PBS, 0.05% (vol/vol) Tween-20) for 5 min. For long term storage, the specimens

were dehydrated in a series of ethanol dilutions and stored in 70% ethanol at 4°C. For whole-

mount immunofluorescence, the samples were permeabilized in Dent's fixative (80% methanol,

20% DMSO) for 15 minutes and followed by rehydration through series of methanol dilutions

(70%, 50% and 25% methanol) for 5 minutes per dilution. The samples were washed with

PBST for 3 x 10 min, then blocked in 5% BSA in PBST for 1 hr at RT. Primary antibody (1:500

dilution, mouse Anti-α-Tubulin Cat # T9026, Sigma-Aldrich) incubation was performed in a

blocking solution (1% BSA in PBST) for 24-36 hr at 4°C. The samples were washed with

PBST for 5 x 5 min, after which samples were incubated with secondary antibodies (1:250

dilution; Goat anti-Mouse IgG Alexa Fluor 594 Cat # A-11032, ThermoFisher) diluted in

blocking solution for overnight at 4°C. Then, the samples were washed with PBST for 5 x 10

min and clarified following iDISCO protocol [45]. Imaging was performed on Leica TCS SP8

DLS and Leica DMi8 confocal microscopes.

Fixation of regenerating specimens for Scanning Electron Microscopy (SEM)

The specimens were fixed overnight at 4°C by 2.5% glutaraldehyde in PBS buffer, then rinsed

thrice with PBS buffer for 10 min. The specimens were postfixed in 1% osmium tetroxide in

PBS buffer at room temperature. After post-fixation, the specimens were dehydrated in a series

of ethanol dilutions until 100% ethanol and absolute ethanol was gradually exchanged with

hexamethyldisilazane (HMDS). After the final 100% HMDS solution step, the specimens were

left in a fume hood overnight to allow HMDS to evaporate. After overnight drying, the samples

were mounted on aluminum stabs and gold coated. SEM imaging was done on JEOL 6610 LV

microscope.



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RNA sequencing and differential gene expression

The regeneration is semi-synchronous among individuals, even though the variation is

considerably mild (generally varies ±1 day). Keeping this in mind we collected the regenerating

samples principally based on morphological signatures apart from the day 1 and 2 samples. For

RNA isolation, the early regenerating samples were collected at days 1 and 2. For later stages,

the samples were primarily collected based on the morphological state extending between day

3-4, day 6-8, day 10-14, day 16-18 (PJUV) and day 21-24 (JUV). Each sample was pooled

from a minimum of three individuals and also included samples from different batches as

detailed in table 1, 1st column. The samples were carefully collected using a pipette and excess

media was removed before snap freezing in liquid nitrogen and stored at -80°C until further

processing. Due to the sheer size of primmorphs at early stages (day 1 and 2) samples collected

from multiple biological replicates were combined to acquire an adequate amount of RNA for

sequencing. For the later stages, only 3 biological replicates were combined into one sample

during RNA isolation. Total RNA was isolated using the TRI Reagent® according to the

manufacturer’s protocol. RNA quality was assessed using Agilent RNA 6000 Nano Kit on

Agilent 2100 Bioanalyzer (Agilent, USA), and only samples with RNA integrity number (RIN)

≥ 8.0 were considered for sequencing. Sample library preparation for RNA sequencing was

accomplished using the SENSE mRNA-Seq Library Prep Kit (Lexogen GmbH). Before

sequencing, the libraries were pre-assessed by Agilent High Sensitivity DNA Kit (Agilent,

USA) and quantified using Qubit™ 1X dsDNA HS Assay Kit (Invitrogen™). The sequencing

was outsourced (GENEWIZ Illumina NovaSeq/HiSeq 2x150 bp sequencing), generating a

20 million paired-end reads per replicate. Raw data was deposited at NCBI GEO submission

GSE149471. After de-multiplexing and filtering high-quality sequencing reads, the adapter

contamination was removed by using Trimmomatic v0.36 [46]. The quality of the reads was

verified using FastQC [47]. Processed reads from each sample were mapped to the S. ciliatum

genome (indexed bowtie2 [48]) by using HISAT2 [49]. Next, the mapped reads were passed

to StringTie [50] for transcript assembly. After initial assembly, all assembled transcripts from

regeneration and development were merged by using StringTie's merge module, which merges

all the gene structures found in any of the samples. The BAM file and GFT file generated by

StringTie's merge were fed into featureCounts [51] to extract the reads counts per transcript.

Differential expression analyses were performed using DESeq2 (Galaxy Version 2.11.40.6)

[52]. Gene models that did not have >10 counts in at least 25% of the samples were excluded.

The principal component analysis (PCA), hierarchical clustering (HC), and heat maps were



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generated using the R package in R-studio (Version 1.2.5019). For functional annotation, the

transcripts were extracted from the GTF annotation file by gffread utility and the LongOrfs

were acquired using TransDecoder. We used blastp [53] with the default curated gathering

threshold to predict the protein orthologues against the Uniprot database. The Gene Ontology

(GO) term enrichment was performed using gene annotation tools including PANTHER™

Classification System [54] and DAVID [55].

Acknowledgments

The authors would like to thank John Bishop and Christine Wood for their support in acquiring

S. ciliatum specimens. We thank Glenn Harper and the team at the Plymouth Electron

Microscopy Lab for their assistance during the imaging. We thank Ro Allen for his help with

R statistics. This work was supported by the Anne Warner endowed Fellowship through the

Marine Biological Association of the UK.



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Figure 1: Schematic representation of embryonic and postlarval development stages in

Sycon ciliatum: depicted based on Leininger, S., et al. 2014, Eerkes-Medrano, D. I. and S.

P. Leys 2006. From left to right: oocyte, cleavage, pre-inversion, post-inversion, amphiblastula

(swimming larva), flat post larva, solid post larva, hollow post larvae, and choanocyte chamber

and asconoid juvenile.



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Figure 2: Morphological and cellular events during the primmorphs stage. The

regeneration proceeds with the aggregation of dissociated cells and transforming the loosely

connected external cells to an intact single layer of pinacocytes. (A-C) Initial multicellular

aggregates in the culture (24 hpd), loosely aggregated primmorphs, the primmorphs have no

defined surface epithelium and made of diverse cell types. (D-F) A general view of primmorphs

in the culture after 2 dpd, (E & F) an intact single layer of cells surrounding primmorphs,

separating from inner cell mass, similar to blastocyst. (G & H) The external ameboid or

cuboidal cells transformed into flat pinacocytes, the external cavity (*) resembles the

extraembryonic cavity with a single layer of flat cells. (E, F, H, I) Fusing the external cells

surrounding the primmorphs, accompanied by the transdifferentiation of the choanocytes and

other external ameboid cells. (J-L) choanocytes transdifferentiation into pinacocytes: (J)



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choanocytes with intact flagella and microvilli, (K & L) gradually these morphological features

were lost through transdifferentiation and forms falt later of pinacocytes. Images are taken with

(A, D, G) bright field, (B, E, H) confocal, (C, F, I) SEM microscope. P: pinacocyte; C:

choanocytes; F: flagella; M: microvilli; (*): external cavity.



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Figure 3: Specule formation during regeneration. (A-F) general view of primmorphs

developing spicules (4-10 dpd). (D & G) Primmorphs developing multiple chambers

surrounded by ciliated cells, simultaneously the spicules start appearing as monaxons. (G-I)

expansion and integration of choanocyte chambers and eventually developing into choanoderm

with a single layer of choanocytes surrounding the atrium. (F & I) note that ostium and osculum

are not formed yet. Images are taken with (A, B, C) bright field, (D, E, F) SEM microscope,

(G, H, I) confocal. C: choanocytes; S: sclerocytes; P: pinacocyte; Mo: monaxons. (*): external

cavity.



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Figure 4: Developing a functional juvenile. (A, B) General view of pre-juvenile and juvenile

stages. (C-J) Osculum opening at apical end and multiple porocytes form ostia. (C) The

appearance of ostium and osculum is synchronized. (G) Ostia in the regenerated juvenile, (H)

a view of ostia from inside, ostia is surrounded by choanocytes. O: Ostia; PO: porocytes; C:

choanocytes; S: sclerocytes ; P: pinacocyte; Os: osculum; A: archaeocytes or amoebocytes.



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Table 1: Summary of the morphological and cellular events during regeneration in

S.ciliatum. The table details various regeneration steps and their overlaps with postlarval

development. 4th column (postlarval development) depicts the morphological events at

postlarval development of S. ciliatum. 1st column details number of samples collected for RNA-

sequencing at each stage of regeneration, N=number of pooled regenerating structures per well,

n=number of pooled regenerating structures per replicate, R=number of replicates per stage.



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Figure 5: A global overview of postlarval and regeneration transcriptomes. (A) Principal

component analysis (PCA) of postlarval datasets, samples at SL, S1, S2, S3, S4, S5, and YS

stages. (B) PCA of regeneration dataset, samples at dpd 1, 2, 3-4, 6-8, 10-14, 16-18 (PJUV)

and 21-24 (JUV). (C & D) hierarchically clustering (HC) on postlarval development (C) and

regeneration (D) datasets revealed two major clusters that we classified as early and late stages.

This analysis confirmed that clusters in regeneration are organized in a hierarchy consistent

with what we observed in the PLD.



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Figure 6: Global transcriptomic comparison of the regeneration and PLD samples. (A)

PCA of larval development versus regeneration datasets. Regeneration exhibits similar

transcriptomic variation as embryogenesis. The regeneration samples collected at D10-14,

PJUV and JUV stages are close to the S5 and PY stages of PLD. (B) Hierarchical clustering,

the dendrogram of both datasets obtained with pvclust is shown with the bootstrap values on

each node. The dendrogram shows a significant correlation among late stages of regeneration

(D10-14, PJUV and JUV) with S5 and PY stages of PLD. (C) Comparison of differentially

expressed genes between PLD and regeneration datasets, similarity matrix for significantly

differentially expressed genes (padj (FDR) < 0.01 for any timepoint comparison against Day 1

for regeneration and S1 for PLD). The yellow box highlighting the similarity between late

stages of regeneration (D10-14, PJUV and JUV) with S5 and PY stages of PLD. (D) The

number of differentially expressed genes in regeneration is highly comparable to PLD and

~50% of genes are commonly expressed.



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Figure 7: Regeneration gene clusters overlapping with PLD gene clusters. A total of 4468

commonly expressed genes between regeneration (A) and PLD (B) are subjected to Fuzzy c-



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means clustering. Based on their gene expression profile we generated 7 clusters in both

datasets. (C) Hierarchical clustering. The dendrogram of all gene expression clusters from

regeneration and PLD. Majority of regeneration gene modules clustered with PLD gene

modules. (D) Cluster overlap table of regeneration versus PLD gene expression clusters

produced via Pearson correlation analysis. Bold text indicates the best cluster overlap and their

matching gene expression profiles are presented on the right side of the table.



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Figure 8: Gene expression across S. ciliatum regeneration. Heatmap of significantly DEG.

(A) A heatmap displaying a selected list of transcriptional factors and Wnt and Tgfb pathways

genes. The majority of listed genes are well known to associate with a range of eumetazoan

developmental regulatory pathways and were previously identified in Sycon ciliatum

embryonic development. (B) A heatmap showing relative expression of ECM and cell adhesion

and motility (integrin, cadherin, and scaffold) genes, a broad range of genes started expressing



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within 24 hpd, possibly involved in cell-aggregation, adhesion, and motility during

regeneration. Heatmap of scaled expression (row z-score) of integrins with significantly

different expression (padj (FDR) < 0.01 for any timepoint comparison against Day 1).



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Figure 9: The origin of animal multicellularity. (A-C) Morphological similarities between

choanocytes and choanoflagellates. (A) S. ciliatum choanocytes among dissociated cells; (B)

choanocyte chambers of sponge S. ciliatum; (C) unicellular and multicellular (colonial) forms

of choanoflagellates. (D) Phylogenetic tree presenting animals and closely related unicellular

holozoan lineages. The position of Ctenophora and Porifera is indicated as a polytomy due to

current uncertainty on their evolutionary origin. Animal ECM and cell-adhesion family

proteins found in unicellular holozoan lineages; empty boxes indicate absence.



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Supplementary File 1: 4D and Z-stack of imaging of primmorphs after 24 hrs post

dissociation.

Supplementary File 2: 4D and Z-stack of imaging of primmorphs 2 days post dissociation.

Supplementary File 3: 4D and Z-stack of imaging of primmorphs 3 days post dissociation.

Supplementary File 4: 4D and Z-stack of imaging of structures with ciliated chambers 8

days post dissociation.

Supplementary File 5: 4D and Z-stack of imaging of regenerating structure at 12 days

post dissociation. Defined choanoderm with a single layer of choanocytes surrounding the

atrium. Note that ostium and osculum are not formed yet.

Supplementary File 6: 4D and Z-stack of imaging of pre-juvenile 18 days post

dissociation. The ostium and osculum are formed.

Supplementary File 7: 4D and Z-stack of imaging of juvenile 24 days post dissociation.



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Supplementary File 8: Differentially expressed genes associated with cell death. (A)

Heatmap shows the relative expression of genes associated with cell death (apoptosis signaling

pathway, P38 MAPK, and response to oxidative stress). The selected list of genes is

significantly DE across the course of S. ciliatum regeneration. Note the large changes in gene

expression at Day 1 and the gradual decrease in the expression later on.



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Regeneration in sponge Sycon ciliatum mimics postlarval … · 2020. 5. 4. · Sycon cil iatum, a calcareous sponge , is widely explored in embryonic and regeneration studies [8,